Immunoassay techniques are widely used for a variety of applications as described in “Quantitative Immunoassay: A Practical Guide for Assay Establishment, Troubleshooting and Clinical Applications; James Wu; AACC Press; 2000”. The most common immunoassay techniques are 1) non-competitive assay: an example of such is the widely known sandwich immunoassay, wherein two binding agents are used to detect an analyte; and 2) competitive assay: wherein only one binding agent is required to detect an analyte.
In its most basic form, the sandwich immunoassay (assay) can be described as follows: a capture antibody, as a first binding agent, is coated (typically) on a solid-phase support. The capture antibody is selected such that it offers a specific affinity to the analyte and ideally does not react with any other analytes. Following this step, a solution containing the target analyte is introduced over this area whereby the target analyte conjugates with the capture antibody. After washing the excess analyte away, a second detection antibody, as a second binding agent, is added to this area. The detection antibody also offers a specific affinity to the analyte and ideally does not react with any other analytes. Furthermore, the detection antibody is typically “labeled” with a reporter agent. The reporter agent is intended to be detectable by one of many detection techniques such as optical (fluorescence or chemiluminescence or large-area imaging), electrical, magnetic or other means. In the assay sequence, the detection antibody further binds with the analyte-capture antibody complex. After removing the excess detection antibody; finally the reporter agent on the detection antibody is interrogated by means of a suitable technique. In this format, the signal from the reporter agent is proportional to the concentration of the analyte within the sample. In the so called “competitive” assay, a competing reaction between detection antibody and (detection antibody+analyte) conjugate is caused. The analyte, or analyte analogue is directly coated on the solid phase and the amount of detection antibody linking to the solid-phase analyte (or analogue) is proportional to the relative concentrations of the detection antibody and the free analyte in solution.